The physiological properties of renal epithelia are intimately related to the regulated, polarized insertion of membrane proteins in highly-specialized cell types that perform unique transport functions along the urinary tubule. These processes depend on the correct addressing, targeting and delivery of subsets of transport vesicles that associates with a variety of accessory proteins to determine selectivity and specificity. The work proposed will focus on collecting duct intercalated cells, which insert Hplus ATPase apically or basolaterally, and provide a unique opportunity to examine protein trafficking pathway s that may be of general cell biological significance. Furthermore, exo-and endocytosis of Hplus ATPase-coated vesicles in intercalated cells occurs by an as yet undefined mechanism. We hypothesize: A) that vesicle transcytosis is responsible for the modulation of apical versus basolateral membrane insertion of Hplus ATPase in intercalated cell subtypes. Specific Aim1 will map intracellular pathways of Hplus ATPase trafficking in A- and B-intercalated cells; B) that Hplus ATPase- transporting vesicles contain 'coat' proteins that modulate the targeting pathways examined in Aim 1. Specific Aim 2 will characterize the accessory proteins associated with trafficking of these vesicles; C) that the Hpluse ATPase complex plays a general role in protein trafficking b regulating the assembly of other vesicle coat proteins (e.g., betaCOP, ARF) that are critical to vesicle fission and fusion, and that the Hplus ATPase is itself regulated by GTP-binding proteins. Specific Aim 3 will examine the role of vesicle pH, Hplus ATPase, and G-proteins in regulating coat assembly and protein trafficking. The aim will take advantage of newly-developed transfected cell culture systems that maintain constitutive and regulated expression of membrane proteins, as well as cell lines that inducibly overexpress heterotrimeric G-proteins. Finally, cells stably transfected with green-fluorescent protein constructs will be used to examine the regulation of membrane trafficking events in real time. These studies on novel vesicle trafficking mechanisms that are amplified in intercalated cells may lead to the identification of sorting mechanisms that are of general relevance to the regulation and dysregulation of transport processes in all cell types.